Telomeres are the deoxyribonucleoprotein structures found at the ends of eukaryotic chromosomes. They are composed of repetitive tracts of duplex G-rich DNA ending in a 3'-single-stranded overhang. Proteins specific to telomeres bind both the double and single-stranded regions to form a distinct structure that functions to protect chromosomes from degradation and end-to-end fusion, to mediate chromosome segregation, and the modulate the replicative potential of the cell by acting as substrates for the enzyme telomerase. Telomeric replication and the regulation of telomere length are currently under intense study because of the role telomeres play in tumorigenesis, the immortalization of human cell lines, and human aging. Cdc13p is a single-stranded telomeric DNA-binding protein identified genetically from the budding yeast Saccharomyces cerevisiae. It is an essential protein which protects the ends of telomeres from degradation and acts as both a positive and negative regulator of telomerase activity. In this study, the single-stranded telomeric DNA recognition activity of the yeast protein Cdc13p will be probed in detail using both biochemical and biophysical approaches. To date, few proteins have been studied that bind single-stranded DNA with sequence specificity, and little is known about the molecular interactions that govern recognition. The aims of our proposal are to: (1) Biochemically probe single-stranded telomeric DNA affinity and specificity of the Cdc13 DNA-binding domain; (2) Use crosslinking and mutagenesis to localize the sites of protein/DNA interaction and use this knowledge for the design of mutants for in vivo studies (3) Determine the solution structures of free protein and its DNA complex using high-resolution heteronuclear NMR spectroscopy. Understanding the molecular basis of the recognition of single- stranded DNA has important implications not only in the field of telomere replication and regulation, but for many other cellular processes involving single-stranded DNA such as transcription, replication, repair, and homologous recombination.